Aim 1. To investigate biologic consequences of BDNF activation of TrkB on chemoresistance. Inhibition of AKT pathway in neuroblastoma inhibits tumor cell growth in vitro and in vivo. Our previous studies have identified activation of the PI3Kinase/Akt/GSK3 pathway mediates resistance to chemotherapy in neuroblastoma cells. Moreover our genetic and pharmacologic studies indicated that activation of AKT alone attenuated the effects of chemotherapy in neuroblastoma cells. Since activated AKT is more highly expressed in tumors of Neuroblastoma patients with a poor prognosis, we screened a number of inhibitors of the AKT that could enhance the efficacy of chemotherapy in our pre-clinical in vitro models. We focused on the AKT inhibitor, Perifosine, because it is Phase I/II trials in adult cancers, with a toxicity profile that is controllable with systemic therapy. Perifosine was tested in a number of our pre-clinical in vitro models as well as our in vivo murine heterotypic and orthotopic xenograft models. Perifosine inhibits activation of AKT and more impressively inhibits tumor cell growth in all 4 cell line models tested in vitro and in vivo. In the AS neuroblastoma model system there was complete tumor regression. In the least sensitive model, the addition of chemotherapy with Perifosine caused dramatic tumor regressions. Moreover Perifosine inhibited the growth of NB tumors containing ALK mutations, even those with the ALKF1174L mutation, which are less sensitive to some small molecule inhibitors of ALK. These studies provide proof of principle that targeting Akt alone will inhibit neuroblastoma cell growth and will synergize with cytotoxics such as etoposide. Recently the Pediatric Phase I study of Perifosine showed activity in neuroblastoma. Based on our pre-clinical study and the Phase I results the FDA approved Perifosine for Orphan Drug Status for the treatment of Neuroblastoma.
Specific Aim 2. To investigate biologic consequences of BDNF activation of TrkB on metastasis and angiogenesis Our previous studies have identified that activation of TrkB stimulated increases in HIF-1a and production of VEGF in Neuroblastoma. Moreover we have found that increases in TrkB stimulate increased invasiveness. In order to model this in vivo additional neuroblastoma Tet-regulated TrkB expression models have been generated so that the cell lines contain the spectrum of genetic alterations found in poor prognosis neuroblastoma tumors. Moreover, we have developed in vitro models that contain more physiologically relevant levels of O2 and evaluated how hypoxia affects the sensitivity of neuroblastoma cells to a number of biologic response modifiers. While it is well established that hypoxia increases VEGF levels, there is only one report indicating that over-expression of TrkA suppresses VEGF via a ligand independent mechanism in NB cells. However, the latter finding is consistent with low levels of VEGF in TrkA-expressing, good prognosis NB tumors. Our interest in regulation of VEGF stemmed from observations noted in preclinical studies where there was a dramatic decrease in VEGF expression and vascularity in tumors of animals receiving either Gleevec or an histone deacetylase inhibitor, first described for MS-27-275 and more recently extended to include Romidepsin. Previously, we detailed that a signal transduction pathway by which IGF activation of IGFR or BDNF activation of TrkB increases VEGF production in NB cells is via PI-3kinase/Akt/mTOR activation of HIF-1a that stimulates increases in VEGF mRNA transcription and translation. Using isogenic Tet-regulated TrkA expressing NB cells43, we confirmed that in the absence of NGF, elevated TrkA levels decreased VEGFmRNA and protein levels decrease. However we noted that in the presence of ligand, activation of TrkA stimulated the PI-3kinase/Akt/mTOR pathway leads to HIF-1a production and stimulation of VEGF mRNA transcription and protein secretion. Our finding that a similar, cognate ligand induced mechanism of induction of VEGF occurred in normal primary cultures of TrkB-expressing cerebellar granule neurons and TrkA-expressing dorsal root ganglion neurons indicates this is a normal not pathologic mechanism by which neurotrophins stimulate VEGF production. Moreover it also provides a mechanism that may account for the normal role neuronally derived VEGF plays in blood vessel development.
Specific Aim 3. Develop in vivo model systems to test targeted agents to the BDNF/TrkB signal transduction pathway The development of a an in vivo pre-clinical murine model of chemoresistance for neuroblastoma. Having identified the TrkB signal transduction pathway as a mediator of chemoresistance in NB cell lines in vitro, we developed an isogenic TET-suppressible TrkB expressing NB tumor cell murine xenograft model for in vivo testing. Mice receiving TET in their drinking water/or food have 3-fold lower levels of TrkB expression in tumors and have increased survival compared to mice with tumors expressing high levels of TrkB (p=0.0003). This aspect of the xenograft model recapitulates the finding that patients whose tumors have elevated TrkB expression have a worse outcome. In our model, we find that the growth of xenograft tumors expressing low levels of TrkB is inhibited by etoposide while the tumors expressing high levels of TrkB continue to grow. The high TrkB expressing tumors expressed elevated levels of activated Akt. Treating mice with a dose of Perifosine that is sufficient to inhibit activated Akt in tumors but does not significantly inhibit tumor growth as a single agent now sensitized the high TrkB expressing tumors to the dose of etoposide to which they were initially resistant. These findings indicate that targeting activated Akt in NB tumors increases their sensitivity to cytotoxic therapy. While studies have shown that it is reasonable to pursue inhibition of the TrkB receptor as an adjunct to therapy, the use of an Akt inhibitor would be valuable in combination or following receptor targeted therapy since targeting receptors may cause activation of AKT due to feedback suppression. Moreover, targeting AKT inhibits a key survival signaling node used not only by TrkB but by other membrane receptors shown to enhance NB cell survival such as IGF1R, IL-6 receptors and VEGFR. By targeting a common downstream signal intermediary, one covers a broader spectrum of signaling pathways that use this common intermediary to affect biologic function.

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National Cancer Institute (NCI)
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Hua, Zhongyan; Zhan, Yue; Zhang, Simeng et al. (2018) P53/PUMA are potential targets that mediate the protection of brain-derived neurotrophic factor (BDNF)/TrkB from etoposide-induced cell death in neuroblastoma (NB). Apoptosis 23:408-419
Gu, Xiao; Hua, Zhongyan; Dong, Yudi et al. (2017) Proteome and Acetylome Analysis Identifies Novel Pathways and Targets Regulated by Perifosine in Neuroblastoma. Sci Rep 7:42062
Odate, Seiichi; Veschi, Veronica; Yan, Shuang et al. (2017) Inhibition of STAT3 with the Generation 2.5 Antisense Oligonucleotide, AZD9150, Decreases Neuroblastoma Tumorigenicity and Increases Chemosensitivity. Clin Cancer Res 23:1771-1784
Li, Zhijie; Zhang, Yi; Tong, Yuxin et al. (2015) Trk inhibitor attenuates the BDNF/TrkB-induced protection of neuroblastoma cells from etoposide in vitro and in vivo. Cancer Biol Ther 16:477-83
Yan, Shuang; Li, Zhijie; Thiele, Carol J (2013) Inhibition of STAT3 with orally active JAK inhibitor, AZD1480, decreases tumor growth in Neuroblastoma and Pediatric Sarcomas In vitro and In vivo. Oncotarget 4:433-45
Thiele, Carol J; Cohn, Susan L (2012) Genetically InFormed therapies--a ""GIFT"" for children with cancer. Clin Cancer Res 18:2735-9
Li, Zhijie; Yan, Shuang; Attayan, Navid et al. (2012) Combination of an allosteric Akt Inhibitor MK-2206 with etoposide or rapamycin enhances the antitumor growth effect in neuroblastoma. Clin Cancer Res 18:3603-15
Nakamura, Katsuya; Tan, Fei; Li, Zhijie et al. (2011) NGF activation of TrkA induces vascular endothelial growth factor expression via induction of hypoxia-inducible factor-1?. Mol Cell Neurosci 46:498-506
Li, Zhijie; Oh, Doo-Yi; Nakamura, Katsuya et al. (2011) Perifosine-induced inhibition of Akt attenuates brain-derived neurotrophic factor/TrkB-induced chemoresistance in neuroblastoma in vivo. Cancer 117:5412-22
Li, Zhijie; Tan, Fei; Liewehr, David J et al. (2010) In vitro and in vivo inhibition of neuroblastoma tumor cell growth by AKT inhibitor perifosine. J Natl Cancer Inst 102:758-70

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